Abstract Optimal mechanical loading is critical for joint tissue health, yet it remains unclear how to optimize loading following knee injury to prevent posttraumatic osteoarthritis (PTOA). The investigators' long-term goal is to develop individualized intervention strategies that target mechanical joint loading for the purpose of preventing PTOA. The objective of this R21 is to determine the acute effect of manipulating mechanical loading, using a real-time feedback paradigm to cue high-loading, low-loading and symmetrical loading conditions, on biomechanical, biochemical and structural measures related to PTOA pathogenesis in humans with anterior cruciate ligament reconstruction (ACLR). Recent evidence from the investigators' laboratory suggests that low- loading following injury may result in deleterious tissue metabolism and worse outcomes. Accordingly, the central hypothesis of this study is that the high-loading condition will result in greater tibiofemoral contact forces but also more knee excursion (less knee stiffness), resulting in a lesser immediate and delayed serum Cartilage Oligomeric Matrix Protein (COMP) response, as well as lesser cartilage deformation. The proposed study is needed because there is growing evidence, contrary to conventional theory, suggesting that patients with lesser mechanical loading following injury may be at higher risk for PTOA, and an intervention that cues higher-loading may be essential in mitigating that risk. Our study will demonstrate how manipulating mechanical load influences multiple critical outcomes needed to understand the mechanistic links between knee biomechanics, COMP response, and cartilage deformation. The central hypothesis will be tested with these two specific aims: 1) determine the acute effects of high-loading, low-loading, and symmetrical loading of the lower extremity on knee kinematics, knee kinetics and tibiofemoral joint contact forces during walking; 2) determine the effects of high-loading, low-loading and symmetrical loading during walking on serum COMP concentrations (immediately post and 3.5 hours post loading) and immediate sonography outcomes of femoral cartilage deformation. The proposed work is innovative as it: 1) incorporates a highly novel combination of outcome measures (biomechanical, joint tissue metabolism, and ultrasound of cartilage structure) that will lead to unprecedented mechanistic insight into the pathogenesis of PTOA; 2) strategically manipulates mechanical loading in multiple directions to determine how a known change in mechanical load will acutely influence outcomes, which is a stark departure from previous cross-sectional studies; and 3) utilizes a novel real-time feedback paradigm to cue changes in mechanical joint loading, which can easily be further developed into a future intervention. This R21 is significant, as the results will be pivotal in understanding how low-loading, high-loading and symmetrical loading influence changes in biomechanical, biochemical and cartilage deformation outcomes, which will lead to unprecedented mechanistic insight into PTOA pathogenesis and provide critical information regarding how best to direct loading following injury to prevent PTOA onset.